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Angiogenic growth factors correlate with disease severity in young patients with autosomal dominant polycystic kidney disease
original article
http://www.kidney-international.org & 2011 International Society of Nephrology
Angiogenic growth factors correlate with disease severity in young patients with autosomal dominant polycystic kidney disease Berenice Y. Reed1, Amirali Masoumi1, Elwaleed Elhassan1, Kim McFann1,2, Melissa A. Cadnapaphornchai3,4, David M. Maahs3,5, Janet K. Snell-Bergeon3,5 and Robert W. Schrier1 1
Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Denver, Aurora, Colorado, USA; Colorado School of Public Health, Biostatistics and Informatics, University of Colorado Denver, Aurora, Colorado, USA; 3Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, Colorado, USA; 4The Children’s Hospital, Aurora, Colorado, USA and 5Barbara Davis Center for Childhood Diabetes, Aurora, Colorado, USA
2
Renal cysts, pain, and hematuria are common presentations of autosomal dominant polycystic kidney disease (ADPKD) in children. Renal function, however, is typically preserved in these patients despite increased renal volume. Since angiogenesis has been implicated in promotion of renal cyst growth in ADPKD, we measured the serum level of various angiogenic factors and early renal structural changes and cardiovascular parameters in 71 patients with ADPKD, with a mean age of 16 years. Renal structure and left ventricular mass index were measured by magnetic resonance imaging or by echocardiogram. Renal function was assessed by creatinine clearance and urinary protein excretion. Serum growth factor levels were measured by enzyme-linked immunosorbent assay. Because of skewed distributions, the various parameters are reported as log10. Serum log10 vascular endothelial growth factor was positively correlated with renal and cardiac structure, but negatively with creatinine clearance. Serum angiopoietin 1 levels significantly correlated with structural change in both the kidney and the heart and with urinary protein. Thus, the correlation between angiogenic growth factors with both renal and cardiac disease severity is compatible with a possible role for angiogenesis in the early progression of disease in ADPKD. Kidney International (2011) 79, 128–134; doi:10.1038/ki.2010.355; published online 29 September 2010 KEYWORDS: ADPKD; cardiovascular disease; children; kidney volume; VEGF
Correspondence: Berenice Y. Reed, Anschutz Medical Campus, Division of Renal Diseases and Hypertension, Mail Stop C283, Building 500, Room C5000B, 13001 East 17th Place, Aurora, Colorado 80045, USA. E-mail: [email protected] Received 17 September 2009; revised 7 July 2010; accepted 21 July 2010; published online 29 September 2010 128
Autosomal dominant polycystic kidney disease (ADPKD) affects between 1 in 400 and 1 in 1000 individuals in the United States.1 Renal cysts are often evident on ultrasound imaging in children who typically become symptomatic in young adulthood.2–4 However, we have previously shown that hypertensive (95th percentile for blood pressure) and borderline hypertensive (75th–95th percentile for blood pressure) children have a higher left ventricular mass index (LVMI) than children with ADPKD and lower blood pressure.5 Moreover, hypertensive children with ADPKD have higher renal volumes than children with lower blood pressure.6 Thus, careful clinical evaluation and monitoring of children with ADPKD is critical. It is apparent that vascular changes including expansion and remodeling must occur in order to support the massive growth of cysts in ADPKD patients. Evidence for angiogenesis in the form of development of a well-defined vascular capsule around human renal cysts in ADPKD supports this tenet.7,8 Moreover, the identification of angiogenic growth factors including vascular endothelial growth factor (VEGF) in the fluid from both human renal and hepatic cysts7–9 and upregulated expression of angiopoietin (Ang)-1, Ang-2, and their endothelial tyrosine kinase (Tie)-2 receptor in ADPKD cholangiocytes supports a role of angiogenic growth factors in mediating this process.10 VEGF is a central mediator of angiogenesis inducing endothelial cell proliferation, sprouting, and promoting vascular leakiness.11 The actions of the angiopoietins appear to be both cell type-specific and dependent upon the expression level of the respective growth factors. Ang-1 expressed by vascular smooth muscle cells and pericytes activates the Tie-2 receptor resulting in blood vessel stabilization.12 However, overexpression of Ang-1 has been associated with increased sprouting and formation of larger blood vessels in transgenic animal studies.13 In contrast, Ang2 causes destabilization of vessels, increased leakiness, and enables function of other cytokines including VEGF.12 In view of the potential opposing roles of Ang-1 and Ang-2, the Kidney International (2011) 79, 128–134
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BY Reed et al.: Angiogenic growth factors and ADPKD
Table 1 | Clinical and demographic characteristics of study population Parameter Age at evaluation (years) Male (%) Height (cm) Weight (kg) Body surface area (BSA; m2) Hypertension (%) Serum creatinine (mg/dl) Ang-1 (ng/ml) Ang-2 (pg/ml) VEGF (pg/ml) Urinary creatinine (mg/24 h) Urinary protein (g/24 h) Creatinine clearance (ml/min per 1.73 m2) Total renal volume (mm3) Total cyst volume (mm3) Renal cyst count LVMI (g/m2)
N
Mean±s.d.
71 29/71 (41%) 71 71 71 27/71 (38%) 70 71 71 70 68 67 70 61 61 61 66
16.3±4.0
Geometric mean and 95% CI
165±14 66±23 1.71±0.34 0.74±0.22 35.52±21.03 2.35±0.96 2997±5327 1365.35±609.50 0.20±0.28 130.24±29.40 653,795±573,871 430,273±534,914 74.5±42.6 61.81±22.20
0.71 27.57 2.16 107.2 1250.24 0.14 125.02 506,0345 254,174
(0.66–0.76) (22.56–33.69) (1.96–2.39) (42.78–268.6) (1129.1–1384.4) (0.12–0.17) (118.85–139.73) (423,634–604,464) (194,691–331,832)
58.13 (53.29–63.41)
Abbreviations: Ang, angiopoietin; BSA, body surface area; CI, confidence interval; LVMI, left ventricular mass index; VEGF, vascular endothelial growth factor.
Ang-1/Ang-2 ratio may also determine the biological activity14–16 Several recent studies support an emerging role for an imbalance of angiogenic growth factor levels in disease processes including tumor growth, diabetes, chronic kidney disease, and cardiovascular disease.12,17–20 As angiogenesis may be an important process for renal cyst expansion, we hypothesized that the serum level of angiogenic growth factors may be linked to measures of disease progression in young patients with ADPKD. Children and young adults with ADPKD present an ideal study population, as renal cysts are present and actively growing. But the disease is largely uncomplicated by features of advanced renal damage including fibrosis, glomerulosclerosis, and inflammation. RESULTS Clinical and demographic characteristics of the study patients
Table 2 | Relationship of VEGF, Ang-1, Ang-2, and ratio of Ang-1/Ang-2 with renal structure Dependent variable Independent variable
b
s.e.
P-value
0.0511 0.0029 0.00002 0.1611
0.0183 0.0014 0.00003 0.0763
0.0073 0.0448 0.5164 0.0393
Log10 total renal cyst volume 0.0862 Log10VEGF Ang-1 0.004 Ang-2 0.00001 Ang-1/Ang-2 0.2616
0.0273 0.0022 0.00005 0.1242
0.0026 0.0680 0.9039 0.0397
Log10 total cyst number Log10VEGF Ang-1 Ang-2 Ang-1/Ang-2
0.0192 0.0014 0.00003 0.0730
0.3027 0.0789 0.3547 0.0109
Log10 total renal volume Log10VEGF Ang-1 Ang-2 Ang-1/Ang-2
0.0199 0.0025 0.00003 0.1923
A total of 71 young patients with age range of 8–26 years, who were enrolled in clinical studies at the University of Colorado at Denver, and who underwent clinical and radiological examination between 2000 and 2009 were included in the study. The patient characteristics are presented in Table 1. Serum VEGF levels were also assessed in 10 young patients (5 male and 5 female) with type 1 diabetes mellitus (serum hemoglobin A1c (%) 8.88±1.63) matched for age (15.7±2.3 years) and normal renal function (creatinine clearance 111±14 ml/min per 1.73 m2) to the young ADPKD patients. Mean serum VEGF level was significantly lower in the young diabetic patients compared with the ADPKD patients (617±389 in diabetic patients compared with 2997± 5327 pg/ml in ADPKD patients, P ¼ 0.0005).
Figure 1. Subjects with VEGF level 475th percentile also had significantly greater log10 total renal and cyst volumes compared with subjects with VEGF level o50th percentile, after correction for age and sex based on a P-value adjusted by Tukey–Kramer correction for multiple comparisons21 (Figures 2, 3). Serum Ang-1 was significantly positively correlated with log10 total renal volume (P ¼ 0.04) after correction for age, sex, and body surface area (Table 2). There were no significant relationships between renal structural parameters and Ang-2. However, the ratio of Ang-1/Ang-2 was significantly and positively correlated with log10 total renal volume, total cyst volume, and cyst count.
Renal structure
Renal function
Serum VEGF was strongly positively correlated with log10 total renal and cyst volume (Table 2). The linear relationship between log10 VEGF and total renal volume is illustrated in
There were significant correlations between Ang-1 and log10 urinary protein excretion (P ¼ 0.009) after correction for both age and sex (Table 3). The linear relationship is depicted
Kidney International (2011) 79, 128–134
Abbreviations: Ang, angiopoietin; VEGF, vascular endothelial growth factor.
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7 6 5 4 3 2 1 0
5.8
0
1
2 3 Log10 VEGF pg/ml
4
5
Figure 1 | Scatter plot of serum log10 vascular endothelial growth factor (VEGF) and log10 total renal volume. The plot depicts the uncorrected relationship between renal volume and serum VEGF. VEGF pg/ml and renal volume mm3.
Log10 total cyst volume mm3
Log10 total renal volume mm3
original article
P = 0.012
5.7 5.6 5.5 5.4 5.3 5.2 5.1 50 –75th percentile
<50th percentile
> 75th percentile
VEGF
Figure 3 | Total cyst volume expressed by quartile of serum vascular endothelial growth factor (VEGF) level. Cyst volume was adjusted for age and sex. VEGF range/quartile as presented in legend for Figure 2. All P-values were corrected for multiple comparisons by the Tukey–Kramer correction.21
Log10 total renal volume mm3
P =0.046 5.95 5.85 5.75
Table 3 | Relationship of VEGF, Ang-1, Ang-2, and ratio of Ang-1/Ang-2 with renal functional parameters including urinary protein and creatinine clearance
5.65
Dependent variable
5.55 <50th percentile
50–75th percentile
> 75th percentile
VEGF
Figure 2 | Total renal volume expressed by quartile of serum vascular endothelial growth factor (VEGF) level. Renal volume was adjusted for age, sex, and body surface area. VEGF range o50th percentile o440 pg/ml; 50–75th percentile 224–2750 pg/ml; 475th percentile 2950–18,000 pg/ml. All P-values were corrected for multiple comparisons by the Tukey–Kramer correction.21
in Figure 4. There were no significant relationships between serum VEGF or Ang-2 levels and urinary protein excretion. The ratio Ang-1/Ang-2 was significantly correlated with log10 urinary protein excretion (Table 3). In this group of young patients with ADPKD and renal function largely preserved (creatinine clearance mean 130.24 ml/min per 1.73 m2), there was a significant negative relationship between log10 VEGF and log10 creatinine clearance (P ¼ 0.02; Table 3). Subjects with VEGF level 475th percentile had significantly lower creatinine clearance compared with those with VEGF level o50th percentile, after correction for multiple comparisons (Figure 5). No significant relationships between Ang-1, Ang-2, Ang-1/Ang-2, and creatinine clearance were found.
Independent variable Log10 urinary protein excretion Log10VEGF Ang-1 Ang-2 Ang-1/Ang-2 Log10 creatinine clearance Log10VEGF Ang-1 Ang-2 Ang-1/Ang-2
s.e.
P-value
0.0401 0.0042 0.00002 0.2050
0.0209 0.0015 0.00004 0.0815
0.0592 0.0093 0.5658 0.0145
0.0165 0.0010 0.00002 0.0102
0.0070 0.0005 0.00001 0.0291
0.0224 0.3020 0.1676 0.7283
b
Abbreviations: Ang, angiopoietin; VEGF, vascular endothelial growth factor.
with VEGF level 475th percentile had significantly higher LVMI compared with those with VEGF level between 50th and 75th percentile and those with VEGF o50th percentile after correction for age, sex, and correction for multiple comparisons (Figure 7). Likewise, subjects with Ang-1 level 475th percentile had significantly higher LVMI compared with those with Ang-1 level o50th percentile (P ¼ 0.013) after correction for age, sex, and correction for multiple comparisons (data not shown). There were no significant relationships between either Ang-2 level or the ratio Ang-1/ Ang-2 and LVMI. Relationship between VEGF and Ang-1
LVMI
Serum VEGF and Ang-1 were positively correlated with log10 LVMI after correction for age and sex (Pp0.0001 and P ¼ 0.04; Table 4). There was a linear relationship between log10 VEGF and log10 LVMI as shown in Figure 6. Subjects 130
Although the distribution of serum VEGF was highly skewed, log10VEGF was significantly positively correlated with Ang-1 level (r ¼ 0.37, P ¼ 0.0019), but not with Ang-2 or with the ratio Ang-1/Ang-2. Forward selection was used in multiple linear regressions to determine independent effects of Ang-1, Kidney International (2011) 79, 128–134
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Table 4 | Relationship of VEGF, Ang-1, Ang-2, and ratio of Ang-1/Ang-2 with LVMI
0.4 0
Dependent variable
–0.4
Independent variable
–0.8
Log10VEGF Ang-1 Ang-2 Ang-1/Ang-2
–1.2 –1.6 0
20
40
60 80 Ang-1 ng/ml
100
120
Figure 4 | Scatter plot of serum angiopoietin (Ang)-1 and log10 urinary protein excretion. The plot depicts the uncorrected relationship between 24 h urinary protein excretion and serum Ang-1 level. Ang-1 ng/ml and urinary protein g/24 h.
P = 0.002 Log10 creatinine clearance ml/min per 1.73 m2
2.2
b
s.e.
P-value
0.0409 0.0014 0.00000 0.0397
0.0078 0.0007 0.00002 0.0344
o0.0001 0.0407 0.8592 0.2531
Abbreviations: Ang, angiopoietin; LVMI, left ventricular mass index; VEGF, vascular endothelial growth factor.
2.5 Log10 LVMI g/m2
Log10 urinary protein g/24 h
BY Reed et al.: Angiogenic growth factors and ADPKD
P = 0.028
2 1.5 1 0.5 0 0
1
2 3 Log10 VEGF pg/ml
4
5
Figure 6 | Scatter plot of serum log10 vascular endothelial growth factor (VEGF) and log10 left ventricular mass index (LVMI). The plot depicts the relationship between serum VEGF and LVMI. VEGF pg/ml and LVMI g/m2.
2.1
P =0.0001 90
2.0 < 50th percentile
50–75th percentile
>75th percentile
80
LVMI g/m2
VEGF
Figure 5 | Creatinine clearance expressed by quartile of serum vascular endothelial growth factor (VEGF) level. Creatinine clearance was corrected for height and weight and VEGF range/ quartile is presented in legend for Figure 1. All P-values were corrected for multiple comparisons by the Tukey–Kramer correction.21
P =0.0015
70 60 50 40 30 20 10
Ang-2, and VEGF on all dependent variables. Table 5 shows that VEGF was selected over Ang-1 in the regression of total renal volume, total cyst volume, LVMI, and creatinine clearance. However, Ang-1 was selected over VEGF for urinary protein excretion. The correlation of VEGF to Ang-1 and their individual relationships to ADPKD phenotypes suggest a mediation effect of VEGF on Ang-1.
0 <50th percentile
50–75th percentile
> 75th percentile
VEGF
DISCUSSION
Figure 7 | Left ventricular mass index (LVMI) expressed by quartile of serum vascular endothelial growth factor (VEGF) level. LVMI is already adjusted for body surface area, but age and sex were included as covariates. VEGF range/quartile as presented in legend for Figure 1. All P-values were corrected for multiple comparisons by the Tukey–Kramer correction.21
In this study we examined the relationship between the angiogenic growth factors VEGF, Ang-1, and Ang-2 and renal and cardiovascular disease progression in young patients with ADPKD. Significant relationships were observed between levels of VEGF, Ang-1, and Ang-1/Ang-2 and renal structural disease, including total renal volume, total cyst volume, and total cyst number (Table 2). These results are indicative of a potential role for upregulated angiogenesis in early renal cyst progression. Indeed, evidence of angiogenesis has previously
been demonstrated on the surface of renal cysts from patients with ADPKD.7,8 Both human liver and renal ADPKD cyst fluids contain VEGF, and a role for angiogenic growth factors in the growth of liver cysts in ADPKD has previously been demonstrated.6,8 Expression of VEGF, VEGF receptor 1, Ang-1, and Tie-2 are strongly upregulated in liver cholangiocytes from ADPKD
Kidney International (2011) 79, 128–134
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Table 5 | Stepwise linear regression models using forward selection to determine independent effects of Ang-1, Ang-2, and VEGF Dependent variable Partial R2
Model R2
P-value
Log10 total renal volume Age Sex Log10VEGF Ang-1 Ang-2
0.2863 0.1379 0.0723 0.0163 0.0065
0.2863 0.4242 0.4965 0.5128 0.5193
o0.0001 0.0006 0.0068 0.1846 0.4013
Log10 total renal cyst volume Age Sex (male) Log10VEGF Ang-1
0.2722 0.1314 0.0929 0.0070
0.2722 0.4036 0.4966 0.5036
o0.0001 0.0010 0.0026 0.3908
Log10 total cyst number Sex Age Ang-1 Log10VEGF Ang-2
0.0941 0.0698 0.0490 0.0539 0.0150
0.0941 0.1638 0.2128 0.2668 0.2818
0.0181 0.0349 0.0697 0.0513 0.2973
Log10 LVMI Log10VEGF Age Sex
0.3965 0.1446 0.0764
0.3965 0.5412 0.6175
o0.0001 o0.0001 0.0009
Log10 urinary protein excretion Sex 0.1943 Ang-1 0.0989 0.0143 Log10VEGF Ang-2 0.0062
0.1943 0.2932 0.3075 0.3137
0.0002 0.0042 0.2616 0.4607
Log10 creatinine clearance Log10VEGF Ang-2
0.0801 0.1108
0.0224 0.1480
Independent variable
0.0801 0.0308
Abbreviations: Ang, angiopoietin; VEGF, vascular endothelial growth factor. Note that all models included log10 VEGF, Ang-1, Ang-2, and appropriate covariates. Ratio of Ang-1/Ang-2 was not included owing to significant collinearity with Ang-1 and Ang-2. Entries represent a summary of forward selection with variables that remained in the model and the order of selection. Po0.15 was required to remain in the model.
patients,10 and inhibition of the VEGF receptor is known to inhibit liver cyst growth in the pkd2(WS25/) mouse model of ADPKD2, supporting a role for angiogenesis in liver cyst growth.22 However, the current study, to the best of our knowledge, is the first to support a relationship between increased VEGF and angiopoietin with structural renal disease progression in human ADPKD. In view of the potential antagonistic effect of Ang-2 on Ang-1 activity,14–16 the Ang-1/Ang-2 ratio may impact biological activity. We therefore also assessed the affect of the ratio on renal and cardiac disease severity. Ang-1/Ang-2 was significantly related to total cyst volume and notably the only measure significantly, albeit weakly, related to total cyst number (P ¼ 0.01). The serum level of VEGF and Ang-2 also appears to be higher in young ADPKD patients compared with previously reported values for healthy young subjects or those with 132
pathological conditions associated with elevated angiogenic growth factors. In the current study, the mean serum VEGF level was 2997 pg/ml compared with a literature reported mean of 306 pg/ml in healthy children.23 In order to assess whether the elevation in serum VEGF reflected impaired renal clearance due to mild renal functional impairment or loss of renal reserve, we compared serum VEGF levels in young diabetic patients who were matched by age, sex, and renal function with the ADPKD patients. Mean serum VEGF level was significantly lower in the diabetic patients compared with the level in the ADPKD patients. Similarly, Ang-2 level in ADPKD was 2350 pg/ml compared with a literature reported control level of 68 (68–1330) pg/ml.14 Mysliwiec et al.24 reported a serum VEGF level of 312 (0–803) pg/ml in young patients with diabetic retinopathy and a healthy control level of 94 (13–328) pg/ml. This is also lower than the levels observed in ADPKD patients in the current study. It should also be noted that renal function was still within the normal range in the young ADPKD subjects in the current study. Serum angiogenic growth factor levels were also significantly related to renal function. Ang-1 levels were significantly correlated with urinary protein excretion (P ¼ 0.009; Table 3). Previous animal studies have demonstrated a strong time-dependent expression of Ang-1 in glomeruli of a diabetic rat model, and correlation with glomerular volume, glomerular area, and urinary protein.25 Serum VEGF level was correlated with creatinine clearance (P ¼ 0.022; Table 3). This result indicates that serum VEGF may indicate very early renal changes as kidney function is essentially preserved in young patients with ADPKD. The mean creatinine clearance in the current study was 130.24±29.4 ml/min per 1.73 m2 (Table 1). Significant positive correlations between LVMI and Ang-1 and VEGF were also demonstrated in the current study. In particular, log10 transformed VEGF level was strongly related to LVMI after correction for age and sex (Po0.0001; Table 4, Figure 7). This is of particular relevance as patients with ADPKD are at an increased risk for left ventricular hypertrophy.26 Moreover, higher serum VEGF level in young ADPKD patients in the current study was associated with elevated LVMI even in the absence of overt hypertension. We have previously shown increased LVMI in children with ADPKD who are hypertensive or who have borderline hypertension (blood pressure between 75th and 95th percentile for age, height, and sex).5 Identification of children at risk for cardiovascular complications is important because cardiac magnetic resonance imaging and/or echocardiography are not routinely performed on young patients with ADPKD. However, future studies to investigate the role of angiogenic growth factors in the etiology of cardiac damage in ADPKD will be necessary. Previous studies have demonstrated increased plasma and platelet levels of Ang-1 and VEGF in hypertension that decreased with treatment.19 In the current study, 26 subjects were hypertensive (blood pressure X95th percentile for age, height, and sex) and 45 were normotensive. However, there Kidney International (2011) 79, 128–134
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BY Reed et al.: Angiogenic growth factors and ADPKD
was no effect of Ang-1 (odds ratio ¼ 0.993 (0.969–1.018), P ¼ 0.59) and VEGF (odds ratio ¼ 0.765 (0.548–1.068), P ¼ 0.1159) on the presence of hypertension. Moreover, it has previously been shown that hypertensive patients with target organ damage (stroke, myocardial infarction, angina, left ventricular hypertrophy, and mild renal failure) had higher levels of VEGF and Ang-1.20 This report agrees with the findings of our current study and indicates that both Ang-1 and VEGF may have potential roles in the early stages of renal and cardiac damage in ADPKD. In the current study, a relationship between VEGF level and Ang-1 was demonstrated. Furthermore, forward selection in multiple linear regression used to assess independent effects of Ang-1 and VEGF on all dependent variables suggest a mediation effect of VEGF on Ang-1 (Table 5). Stimulation of Ang-1 by VEGF has previously been demonstrated in bovine retinal pericytes.27 In addition, a recent study demonstrated that VEGF activates Tie-2 via proteolytic cleavage of the Tie-1 tyrosine kinase resulting in transphosphorylation and activation of Tie-2.28 This suggests a synergistic action of the angiopoietin and VEGF growth factors. However, while VEGF appeared to have a stronger relationship to renal structural parameters and LVMI, notably each growth factor had a unique relationship between specific renal functional parameters as evidenced by the relationship between VEGF and creatinine clearance and Ang-1 with urinary protein excretion. Activation of the renin–angiotensin system occurs early in ADPKD29–32 and may in part explain the upregulated expression of both VEGF and angiopoietins. In rats, increased renal expression of VEGF, Ang-1, and Ang-2 following infusion of angiotensin has previously been demonstrated.33,34 Furthermore, Ang-2 has been shown to stimulate division of human endothelial cells in culture and to upregulate expression of VEGF.35 These effects were blocked by candesartan implicating involvement of the angiotensin-1 receptor. In conclusion, VEGF level increases with expanding kidney volume and rise in LVMI, possibly reflecting a role of this growth factor in early renal and cardiac disease progression in ADPKD. Future animal and cell studies that focus on angiogenesis in ADPKD models will be required to unravel the involved molecular mechanisms, but may provide potential new therapeutic targets for ADPKD. METHODS Patient description and clinical evaluation
All patients gave informed consent to a protocol that was approved by the Colorado Multiple Institute Review Board. Patients were recruited from those participating in ongoing clinical studies of ADPKD at the University of Colorado Denver between 1990 and 2009. ADPKD diagnosis was based on radiographic imaging and Ravine’s criteria;36 all patients included in this study had a positive family history of ADPKD. All patients were studied during a 2-day in-patient visit at the Pediatric Clinical Translational Research Center at the Children’s Hospital in Denver Colorado, as described Kidney International (2011) 79, 128–134
previously.5 Serum and urine creatinine, height, and weight were used to calculate creatinine clearance for all subjects. Blood was drawn for serum chemistries and growth factor assay. Two 24 h urines were collected for urinary protein excretion and the value reported is the mean for both 24 h urines. Urinary protein was determined in the hospital core laboratory by the pyrogallol red method. Left ventricular mass was determined by standard two-dimensional and Doppler echocardiogram as previously described5 or by cardiac magnetic resonance imaging on a 1.5 T Siemens system (Siemens, Malvern, PA, USA). Cardiac-gated breathhold two dimensional true-Fast Imaging with Steady state Precession (Fast Imaging Employing Steady-state Acquisition) short-axis cine images were obtained to cover the left ventricle atrioventricular ring to apex (10 mm slice thickness, no gap, field of view 250–320 mm; 10–15 breath holds to cover the whole ventricle). The imaging protocol consisted of localizers in three orthogonal planes (axial, coronal, and sagital). After the data were acquired, a Leonardo (Siemens Medical System) workstation was used to manually contour endocardial and epicardial contours of the left ventricle in end-diastole and end-systole, and left ventricular mass was calculated. Left ventricular mass was corrected by body surface area and reported as LVMI in g/m2. Angiopoietin and VEGF assay
Patient sera were stored at –80 1C before assay and all samples were assayed in duplicate. Ang-1 and VEGF were assayed by enzyme-linked immunosorbent assay using kits from Raybiotech (Norcross, GA, USA). The intra- and inter-assay coefficient of variability of the Ang-1 assay was o10% and o12%, respectively, and lower limit of detection 30 pg/ml. The VEGF intra- and inter-assay coefficient of variability was o10 and o12%, respectively, with lower limit of detection 20 pg/ml. Serum Ang-2 was assayed by Quantikine enzyme-linked immunosorbent assay from R&D Systems (Minneapolis, MN, USA). The intra- and inter-assay reproducibility was 7 and 10.4%, respectively, with lower limit of detection, 21.3 pg/ml. Statistical analysis
All variables were checked for the distributional assumption of normality. Owing to the highly skewed distributions of all outcome variables, log10 transformations were applied and the transformed variables were used in all analyses. Log10 transformations were also applied to VEGF and the ratio of Ang-1/Ang-2. All statistical analyses were performed using SAS version 9.2 (SAS Institute, Cary, NC, USA). Descriptive statistics are presented as mean, s.d., and 95% confidence interval or geometric mean and 95% confidence interval. Univariate linear regression was used to test the relationship of VEGF, Ang-1, Ang-2, and the ratio of Ang-1/Ang-2 on ADPKD phenotypes including total renal volume, total cyst number, total cyst volume, creatinine clearance, LVMI, and urinary protein excretion. Stepwise linear regression was used with the forward selection method to determine independent 133
original article
effects of Ang-1, Ang-2, and VEGF on all dependent variables. No correction for multiple end points was applied; however, the primary hypothesis was that Ang-1 would be related to kidney volume measures. In linear regression, renal volume was adjusted for age, sex, and body surface area, renal cyst volume was adjusted for age and sex. Creatinine clearance was corrected for bovine serum albumin, and proteinuria was corrected for age and sex. LVMI was already adjusted for body surface area, but age and sex were included as covariates. Logistic regression was used to test the relationship of VEGF, Ang-1, Ang-2, and ratio Ang-1/Ang-2 with hypertension. In analysis of total renal volume, total cyst volume, creatinine clearance, and LVMI by quartile of VEGF; all P-values were corrected for multiple comparisons by the Tukey–Kramer correction.21
BY Reed et al.: Angiogenic growth factors and ADPKD
13. 14.
15. 16. 17.
18.
19.
20.
DISCLOSURE
All the authors declared no competing interests. 21.
ACKNOWLEDGMENTS
This research was supported by Grant numbers M01RR00051, M01RR00069, the General Research Centers Program, National Center for Research Resources (NCRR)/NIH; by NIH/NCRR Colorado CTSI Grant number ULI RR025780, by Grant DK34039 from NIH (NIDDK), and by the Zell Family Foundation. The content of this publication is the author’s sole responsibility and does not necessarily represent the official NIH views.
22. 23.
24.
25.
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Suri C, McClain J, Thurston G et al. Increased vascularization in mice overexpressing angiopoietin-1. Science 1998; 282: 468–471. Lovegrove FE, Tangpukdee N, Opoka RO et al. Serum angiopoietin-1 and -2 levels discriminate cerebral malaria from uncomplicated malaria and predict clinical outcome in African children. PlosOne 2009; 4: e4912, 2009. Fielder U, Augustin HG. Angiopoietins: a link between angiogenesis and inflammation. Trends Immunol 2006; 27: 552–558. Bolin M, Wiberg-Itzel E, Wilkstrom AK et al. Angiopoietin-1/angiopoietin-2 ratio for prediction of preeclampsia. Am J Hypertens 2009; 22: 891–895. Lim HS, Lip GYH, Blann AD. Angiopoietin-1 and angiopoietin-2 in diabetes mellitus: relationship to VEGF, glycaemic control, endothelial damage/dysfunction and atherosclerosis. Atherosclerosis 2005; 180: 113–118. Sascha D, Kumpers P, Hellpap J et al. Angiopoietin 2 and cardiovascular disease in dialysis and kidney transplantation. Am J Kid Dis 2009; 53: 770–778. Nadar SK, Blann AD, Lip GYH. Plasma and platelet-derived vascular endothelial growth factor and angiopoietin-1 in hypertension: effects of antihypertensive therapy. J Int Med 2004; 256: 331–337. Nadar SK, Blann A, Beevers DG et al. Abnormal angiopoietins 1 & 2, angiopoietin receptor Tie-2 and vascular endothelial growth factor levels in hypertension: relationship to target organ damage [a sub-study of the Anglo-Scandanavian Cardiac Outcomes Trial (ASCOT)]. J Int Med 2004; 258: 336–343. Kramer CY. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics 1966; 12: 307–310. Amura CA, Brodsky KS, Groff R et al. VEGF receptor inhibition blocks liver cyst growth in pkd(WS25/)mice. Am J Cell Physiol 2007; 293: C419–C428. Heshmat NM, El-Kerdany TH. Serum levels of vascular endothelial growth factor in children and adolescents with sytemic lupus erythematosus. Pediatr Allergy Immunol 2007; 18: 346–353. Mysliwiec M, Balcerska A, Zorena K et al. The role of vascular endothelial growth factor, tumor necrosis factor alpha and interleukin-6 in pathogenesis of diabetic retinopathy. Diabetes Care Clin Practice 2008; 79: 141–146. Yang YB, Liu F, Huang SM. The expression and significance of the angiopoietin-1 in the kidney of diabetic rats. Sichuan Da Xue Xue Bao Yi Xue Ban 2007; 38: 93–96. Chapman AB, Johnson AM, Rainguet S et al. Left ventricular hypertrophy in autosomal dominant polycystic kidney disease. J Am Soc Nephrol 1997; 8: 1292–1297. Park YS, Kim NH, Jo I. Hypoxia and vascular endothelial growth factor acutely up-regulate angiopoietin-1 and Tie2 mRNA in bovine retinal pericytes. Microvasc Res 2003; 65: 125–131. Singh H, Milner CS, Aguilar Hernandez MM et al. Vascular endothelial growth factor activates the tie family of receptor tyrosine kinases. Cell Signal 2009; 21: 1346–1350. Chapman AB, Johnson AM, Gabow P et al. The renin-angiotensinaldosterone system and autosomal polycystic kidney disease. N Eng J Med 1990; 323: 1091–1096. Schrier RW. Renal volume, renin-angiotensin-aldosterone system, hypertension, and left ventricular hypertrophy in patients with autosomal dominant polycystic kidney disease. J Am Soc Nephrol 2009; 20: 1888–1893. Barrett BJ, Foley R, Morgan J et al. Differences in hormonal and renal vascular responses between normotensive patients with autosomal dominant polycystic kidney disease and unaffected family members. Kidney Int 1994; 46: 1118–1123. Harrap SB, Davies DL, Macnicol AM et al. Renal, cardiovascular and hormonal characteristics of young adults with autosomal dominant polycystic kidney disease. Kidney Int 1991; 40: 501–508. Rizkalla B, Forbes JM, Cooper ME et al. Increased renal vascular endothelial growth factor and angiopoietins by angiotensin II infusion is mediated by both AT1 and AT2 receptors. J Am Soc Nephrol 2003; 14: 3061–3071. Kitayama H, Maeshima Y, Takazawa Y et al. Regulation of angiogenic factors in angiotensin II infusion model in association with tubulointerstitial injuries. Am J Hypertens 2006; 19: 718–727. Herr D, Rodewald M, Fraser HM et al. Regulation of endothelial proliferation by the renin-angiotensin system in human umbilical vein endothelial cells. Reproduction 2008; 136: 125–130. Ravine D, Gibson RN, Walker RG et al. Evaluation of ultrasonographic diagnostic criteria for autosomal dominant polycystic kidney 1. Lancet 1994; 343: 824–827.
Kidney International (2011) 79, 128–134
http://www.kidney-international.org & 2011 International Society of Nephrology
Angiogenic growth factors correlate with disease severity in young patients with autosomal dominant polycystic kidney disease Berenice Y. Reed1, Amirali Masoumi1, Elwaleed Elhassan1, Kim McFann1,2, Melissa A. Cadnapaphornchai3,4, David M. Maahs3,5, Janet K. Snell-Bergeon3,5 and Robert W. Schrier1 1
Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Denver, Aurora, Colorado, USA; Colorado School of Public Health, Biostatistics and Informatics, University of Colorado Denver, Aurora, Colorado, USA; 3Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, Colorado, USA; 4The Children’s Hospital, Aurora, Colorado, USA and 5Barbara Davis Center for Childhood Diabetes, Aurora, Colorado, USA
2
Renal cysts, pain, and hematuria are common presentations of autosomal dominant polycystic kidney disease (ADPKD) in children. Renal function, however, is typically preserved in these patients despite increased renal volume. Since angiogenesis has been implicated in promotion of renal cyst growth in ADPKD, we measured the serum level of various angiogenic factors and early renal structural changes and cardiovascular parameters in 71 patients with ADPKD, with a mean age of 16 years. Renal structure and left ventricular mass index were measured by magnetic resonance imaging or by echocardiogram. Renal function was assessed by creatinine clearance and urinary protein excretion. Serum growth factor levels were measured by enzyme-linked immunosorbent assay. Because of skewed distributions, the various parameters are reported as log10. Serum log10 vascular endothelial growth factor was positively correlated with renal and cardiac structure, but negatively with creatinine clearance. Serum angiopoietin 1 levels significantly correlated with structural change in both the kidney and the heart and with urinary protein. Thus, the correlation between angiogenic growth factors with both renal and cardiac disease severity is compatible with a possible role for angiogenesis in the early progression of disease in ADPKD. Kidney International (2011) 79, 128–134; doi:10.1038/ki.2010.355; published online 29 September 2010 KEYWORDS: ADPKD; cardiovascular disease; children; kidney volume; VEGF
Correspondence: Berenice Y. Reed, Anschutz Medical Campus, Division of Renal Diseases and Hypertension, Mail Stop C283, Building 500, Room C5000B, 13001 East 17th Place, Aurora, Colorado 80045, USA. E-mail: [email protected] Received 17 September 2009; revised 7 July 2010; accepted 21 July 2010; published online 29 September 2010 128
Autosomal dominant polycystic kidney disease (ADPKD) affects between 1 in 400 and 1 in 1000 individuals in the United States.1 Renal cysts are often evident on ultrasound imaging in children who typically become symptomatic in young adulthood.2–4 However, we have previously shown that hypertensive (95th percentile for blood pressure) and borderline hypertensive (75th–95th percentile for blood pressure) children have a higher left ventricular mass index (LVMI) than children with ADPKD and lower blood pressure.5 Moreover, hypertensive children with ADPKD have higher renal volumes than children with lower blood pressure.6 Thus, careful clinical evaluation and monitoring of children with ADPKD is critical. It is apparent that vascular changes including expansion and remodeling must occur in order to support the massive growth of cysts in ADPKD patients. Evidence for angiogenesis in the form of development of a well-defined vascular capsule around human renal cysts in ADPKD supports this tenet.7,8 Moreover, the identification of angiogenic growth factors including vascular endothelial growth factor (VEGF) in the fluid from both human renal and hepatic cysts7–9 and upregulated expression of angiopoietin (Ang)-1, Ang-2, and their endothelial tyrosine kinase (Tie)-2 receptor in ADPKD cholangiocytes supports a role of angiogenic growth factors in mediating this process.10 VEGF is a central mediator of angiogenesis inducing endothelial cell proliferation, sprouting, and promoting vascular leakiness.11 The actions of the angiopoietins appear to be both cell type-specific and dependent upon the expression level of the respective growth factors. Ang-1 expressed by vascular smooth muscle cells and pericytes activates the Tie-2 receptor resulting in blood vessel stabilization.12 However, overexpression of Ang-1 has been associated with increased sprouting and formation of larger blood vessels in transgenic animal studies.13 In contrast, Ang2 causes destabilization of vessels, increased leakiness, and enables function of other cytokines including VEGF.12 In view of the potential opposing roles of Ang-1 and Ang-2, the Kidney International (2011) 79, 128–134
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Table 1 | Clinical and demographic characteristics of study population Parameter Age at evaluation (years) Male (%) Height (cm) Weight (kg) Body surface area (BSA; m2) Hypertension (%) Serum creatinine (mg/dl) Ang-1 (ng/ml) Ang-2 (pg/ml) VEGF (pg/ml) Urinary creatinine (mg/24 h) Urinary protein (g/24 h) Creatinine clearance (ml/min per 1.73 m2) Total renal volume (mm3) Total cyst volume (mm3) Renal cyst count LVMI (g/m2)
N
Mean±s.d.
71 29/71 (41%) 71 71 71 27/71 (38%) 70 71 71 70 68 67 70 61 61 61 66
16.3±4.0
Geometric mean and 95% CI
165±14 66±23 1.71±0.34 0.74±0.22 35.52±21.03 2.35±0.96 2997±5327 1365.35±609.50 0.20±0.28 130.24±29.40 653,795±573,871 430,273±534,914 74.5±42.6 61.81±22.20
0.71 27.57 2.16 107.2 1250.24 0.14 125.02 506,0345 254,174
(0.66–0.76) (22.56–33.69) (1.96–2.39) (42.78–268.6) (1129.1–1384.4) (0.12–0.17) (118.85–139.73) (423,634–604,464) (194,691–331,832)
58.13 (53.29–63.41)
Abbreviations: Ang, angiopoietin; BSA, body surface area; CI, confidence interval; LVMI, left ventricular mass index; VEGF, vascular endothelial growth factor.
Ang-1/Ang-2 ratio may also determine the biological activity14–16 Several recent studies support an emerging role for an imbalance of angiogenic growth factor levels in disease processes including tumor growth, diabetes, chronic kidney disease, and cardiovascular disease.12,17–20 As angiogenesis may be an important process for renal cyst expansion, we hypothesized that the serum level of angiogenic growth factors may be linked to measures of disease progression in young patients with ADPKD. Children and young adults with ADPKD present an ideal study population, as renal cysts are present and actively growing. But the disease is largely uncomplicated by features of advanced renal damage including fibrosis, glomerulosclerosis, and inflammation. RESULTS Clinical and demographic characteristics of the study patients
Table 2 | Relationship of VEGF, Ang-1, Ang-2, and ratio of Ang-1/Ang-2 with renal structure Dependent variable Independent variable
b
s.e.
P-value
0.0511 0.0029 0.00002 0.1611
0.0183 0.0014 0.00003 0.0763
0.0073 0.0448 0.5164 0.0393
Log10 total renal cyst volume 0.0862 Log10VEGF Ang-1 0.004 Ang-2 0.00001 Ang-1/Ang-2 0.2616
0.0273 0.0022 0.00005 0.1242
0.0026 0.0680 0.9039 0.0397
Log10 total cyst number Log10VEGF Ang-1 Ang-2 Ang-1/Ang-2
0.0192 0.0014 0.00003 0.0730
0.3027 0.0789 0.3547 0.0109
Log10 total renal volume Log10VEGF Ang-1 Ang-2 Ang-1/Ang-2
0.0199 0.0025 0.00003 0.1923
A total of 71 young patients with age range of 8–26 years, who were enrolled in clinical studies at the University of Colorado at Denver, and who underwent clinical and radiological examination between 2000 and 2009 were included in the study. The patient characteristics are presented in Table 1. Serum VEGF levels were also assessed in 10 young patients (5 male and 5 female) with type 1 diabetes mellitus (serum hemoglobin A1c (%) 8.88±1.63) matched for age (15.7±2.3 years) and normal renal function (creatinine clearance 111±14 ml/min per 1.73 m2) to the young ADPKD patients. Mean serum VEGF level was significantly lower in the young diabetic patients compared with the ADPKD patients (617±389 in diabetic patients compared with 2997± 5327 pg/ml in ADPKD patients, P ¼ 0.0005).
Figure 1. Subjects with VEGF level 475th percentile also had significantly greater log10 total renal and cyst volumes compared with subjects with VEGF level o50th percentile, after correction for age and sex based on a P-value adjusted by Tukey–Kramer correction for multiple comparisons21 (Figures 2, 3). Serum Ang-1 was significantly positively correlated with log10 total renal volume (P ¼ 0.04) after correction for age, sex, and body surface area (Table 2). There were no significant relationships between renal structural parameters and Ang-2. However, the ratio of Ang-1/Ang-2 was significantly and positively correlated with log10 total renal volume, total cyst volume, and cyst count.
Renal structure
Renal function
Serum VEGF was strongly positively correlated with log10 total renal and cyst volume (Table 2). The linear relationship between log10 VEGF and total renal volume is illustrated in
There were significant correlations between Ang-1 and log10 urinary protein excretion (P ¼ 0.009) after correction for both age and sex (Table 3). The linear relationship is depicted
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Abbreviations: Ang, angiopoietin; VEGF, vascular endothelial growth factor.
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7 6 5 4 3 2 1 0
5.8
0
1
2 3 Log10 VEGF pg/ml
4
5
Figure 1 | Scatter plot of serum log10 vascular endothelial growth factor (VEGF) and log10 total renal volume. The plot depicts the uncorrected relationship between renal volume and serum VEGF. VEGF pg/ml and renal volume mm3.
Log10 total cyst volume mm3
Log10 total renal volume mm3
original article
P = 0.012
5.7 5.6 5.5 5.4 5.3 5.2 5.1 50 –75th percentile
<50th percentile
> 75th percentile
VEGF
Figure 3 | Total cyst volume expressed by quartile of serum vascular endothelial growth factor (VEGF) level. Cyst volume was adjusted for age and sex. VEGF range/quartile as presented in legend for Figure 2. All P-values were corrected for multiple comparisons by the Tukey–Kramer correction.21
Log10 total renal volume mm3
P =0.046 5.95 5.85 5.75
Table 3 | Relationship of VEGF, Ang-1, Ang-2, and ratio of Ang-1/Ang-2 with renal functional parameters including urinary protein and creatinine clearance
5.65
Dependent variable
5.55 <50th percentile
50–75th percentile
> 75th percentile
VEGF
Figure 2 | Total renal volume expressed by quartile of serum vascular endothelial growth factor (VEGF) level. Renal volume was adjusted for age, sex, and body surface area. VEGF range o50th percentile o440 pg/ml; 50–75th percentile 224–2750 pg/ml; 475th percentile 2950–18,000 pg/ml. All P-values were corrected for multiple comparisons by the Tukey–Kramer correction.21
in Figure 4. There were no significant relationships between serum VEGF or Ang-2 levels and urinary protein excretion. The ratio Ang-1/Ang-2 was significantly correlated with log10 urinary protein excretion (Table 3). In this group of young patients with ADPKD and renal function largely preserved (creatinine clearance mean 130.24 ml/min per 1.73 m2), there was a significant negative relationship between log10 VEGF and log10 creatinine clearance (P ¼ 0.02; Table 3). Subjects with VEGF level 475th percentile had significantly lower creatinine clearance compared with those with VEGF level o50th percentile, after correction for multiple comparisons (Figure 5). No significant relationships between Ang-1, Ang-2, Ang-1/Ang-2, and creatinine clearance were found.
Independent variable Log10 urinary protein excretion Log10VEGF Ang-1 Ang-2 Ang-1/Ang-2 Log10 creatinine clearance Log10VEGF Ang-1 Ang-2 Ang-1/Ang-2
s.e.
P-value
0.0401 0.0042 0.00002 0.2050
0.0209 0.0015 0.00004 0.0815
0.0592 0.0093 0.5658 0.0145
0.0165 0.0010 0.00002 0.0102
0.0070 0.0005 0.00001 0.0291
0.0224 0.3020 0.1676 0.7283
b
Abbreviations: Ang, angiopoietin; VEGF, vascular endothelial growth factor.
with VEGF level 475th percentile had significantly higher LVMI compared with those with VEGF level between 50th and 75th percentile and those with VEGF o50th percentile after correction for age, sex, and correction for multiple comparisons (Figure 7). Likewise, subjects with Ang-1 level 475th percentile had significantly higher LVMI compared with those with Ang-1 level o50th percentile (P ¼ 0.013) after correction for age, sex, and correction for multiple comparisons (data not shown). There were no significant relationships between either Ang-2 level or the ratio Ang-1/ Ang-2 and LVMI. Relationship between VEGF and Ang-1
LVMI
Serum VEGF and Ang-1 were positively correlated with log10 LVMI after correction for age and sex (Pp0.0001 and P ¼ 0.04; Table 4). There was a linear relationship between log10 VEGF and log10 LVMI as shown in Figure 6. Subjects 130
Although the distribution of serum VEGF was highly skewed, log10VEGF was significantly positively correlated with Ang-1 level (r ¼ 0.37, P ¼ 0.0019), but not with Ang-2 or with the ratio Ang-1/Ang-2. Forward selection was used in multiple linear regressions to determine independent effects of Ang-1, Kidney International (2011) 79, 128–134
original article
Table 4 | Relationship of VEGF, Ang-1, Ang-2, and ratio of Ang-1/Ang-2 with LVMI
0.4 0
Dependent variable
–0.4
Independent variable
–0.8
Log10VEGF Ang-1 Ang-2 Ang-1/Ang-2
–1.2 –1.6 0
20
40
60 80 Ang-1 ng/ml
100
120
Figure 4 | Scatter plot of serum angiopoietin (Ang)-1 and log10 urinary protein excretion. The plot depicts the uncorrected relationship between 24 h urinary protein excretion and serum Ang-1 level. Ang-1 ng/ml and urinary protein g/24 h.
P = 0.002 Log10 creatinine clearance ml/min per 1.73 m2
2.2
b
s.e.
P-value
0.0409 0.0014 0.00000 0.0397
0.0078 0.0007 0.00002 0.0344
o0.0001 0.0407 0.8592 0.2531
Abbreviations: Ang, angiopoietin; LVMI, left ventricular mass index; VEGF, vascular endothelial growth factor.
2.5 Log10 LVMI g/m2
Log10 urinary protein g/24 h
BY Reed et al.: Angiogenic growth factors and ADPKD
P = 0.028
2 1.5 1 0.5 0 0
1
2 3 Log10 VEGF pg/ml
4
5
Figure 6 | Scatter plot of serum log10 vascular endothelial growth factor (VEGF) and log10 left ventricular mass index (LVMI). The plot depicts the relationship between serum VEGF and LVMI. VEGF pg/ml and LVMI g/m2.
2.1
P =0.0001 90
2.0 < 50th percentile
50–75th percentile
>75th percentile
80
LVMI g/m2
VEGF
Figure 5 | Creatinine clearance expressed by quartile of serum vascular endothelial growth factor (VEGF) level. Creatinine clearance was corrected for height and weight and VEGF range/ quartile is presented in legend for Figure 1. All P-values were corrected for multiple comparisons by the Tukey–Kramer correction.21
P =0.0015
70 60 50 40 30 20 10
Ang-2, and VEGF on all dependent variables. Table 5 shows that VEGF was selected over Ang-1 in the regression of total renal volume, total cyst volume, LVMI, and creatinine clearance. However, Ang-1 was selected over VEGF for urinary protein excretion. The correlation of VEGF to Ang-1 and their individual relationships to ADPKD phenotypes suggest a mediation effect of VEGF on Ang-1.
0 <50th percentile
50–75th percentile
> 75th percentile
VEGF
DISCUSSION
Figure 7 | Left ventricular mass index (LVMI) expressed by quartile of serum vascular endothelial growth factor (VEGF) level. LVMI is already adjusted for body surface area, but age and sex were included as covariates. VEGF range/quartile as presented in legend for Figure 1. All P-values were corrected for multiple comparisons by the Tukey–Kramer correction.21
In this study we examined the relationship between the angiogenic growth factors VEGF, Ang-1, and Ang-2 and renal and cardiovascular disease progression in young patients with ADPKD. Significant relationships were observed between levels of VEGF, Ang-1, and Ang-1/Ang-2 and renal structural disease, including total renal volume, total cyst volume, and total cyst number (Table 2). These results are indicative of a potential role for upregulated angiogenesis in early renal cyst progression. Indeed, evidence of angiogenesis has previously
been demonstrated on the surface of renal cysts from patients with ADPKD.7,8 Both human liver and renal ADPKD cyst fluids contain VEGF, and a role for angiogenic growth factors in the growth of liver cysts in ADPKD has previously been demonstrated.6,8 Expression of VEGF, VEGF receptor 1, Ang-1, and Tie-2 are strongly upregulated in liver cholangiocytes from ADPKD
Kidney International (2011) 79, 128–134
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Table 5 | Stepwise linear regression models using forward selection to determine independent effects of Ang-1, Ang-2, and VEGF Dependent variable Partial R2
Model R2
P-value
Log10 total renal volume Age Sex Log10VEGF Ang-1 Ang-2
0.2863 0.1379 0.0723 0.0163 0.0065
0.2863 0.4242 0.4965 0.5128 0.5193
o0.0001 0.0006 0.0068 0.1846 0.4013
Log10 total renal cyst volume Age Sex (male) Log10VEGF Ang-1
0.2722 0.1314 0.0929 0.0070
0.2722 0.4036 0.4966 0.5036
o0.0001 0.0010 0.0026 0.3908
Log10 total cyst number Sex Age Ang-1 Log10VEGF Ang-2
0.0941 0.0698 0.0490 0.0539 0.0150
0.0941 0.1638 0.2128 0.2668 0.2818
0.0181 0.0349 0.0697 0.0513 0.2973
Log10 LVMI Log10VEGF Age Sex
0.3965 0.1446 0.0764
0.3965 0.5412 0.6175
o0.0001 o0.0001 0.0009
Log10 urinary protein excretion Sex 0.1943 Ang-1 0.0989 0.0143 Log10VEGF Ang-2 0.0062
0.1943 0.2932 0.3075 0.3137
0.0002 0.0042 0.2616 0.4607
Log10 creatinine clearance Log10VEGF Ang-2
0.0801 0.1108
0.0224 0.1480
Independent variable
0.0801 0.0308
Abbreviations: Ang, angiopoietin; VEGF, vascular endothelial growth factor. Note that all models included log10 VEGF, Ang-1, Ang-2, and appropriate covariates. Ratio of Ang-1/Ang-2 was not included owing to significant collinearity with Ang-1 and Ang-2. Entries represent a summary of forward selection with variables that remained in the model and the order of selection. Po0.15 was required to remain in the model.
patients,10 and inhibition of the VEGF receptor is known to inhibit liver cyst growth in the pkd2(WS25/) mouse model of ADPKD2, supporting a role for angiogenesis in liver cyst growth.22 However, the current study, to the best of our knowledge, is the first to support a relationship between increased VEGF and angiopoietin with structural renal disease progression in human ADPKD. In view of the potential antagonistic effect of Ang-2 on Ang-1 activity,14–16 the Ang-1/Ang-2 ratio may impact biological activity. We therefore also assessed the affect of the ratio on renal and cardiac disease severity. Ang-1/Ang-2 was significantly related to total cyst volume and notably the only measure significantly, albeit weakly, related to total cyst number (P ¼ 0.01). The serum level of VEGF and Ang-2 also appears to be higher in young ADPKD patients compared with previously reported values for healthy young subjects or those with 132
pathological conditions associated with elevated angiogenic growth factors. In the current study, the mean serum VEGF level was 2997 pg/ml compared with a literature reported mean of 306 pg/ml in healthy children.23 In order to assess whether the elevation in serum VEGF reflected impaired renal clearance due to mild renal functional impairment or loss of renal reserve, we compared serum VEGF levels in young diabetic patients who were matched by age, sex, and renal function with the ADPKD patients. Mean serum VEGF level was significantly lower in the diabetic patients compared with the level in the ADPKD patients. Similarly, Ang-2 level in ADPKD was 2350 pg/ml compared with a literature reported control level of 68 (68–1330) pg/ml.14 Mysliwiec et al.24 reported a serum VEGF level of 312 (0–803) pg/ml in young patients with diabetic retinopathy and a healthy control level of 94 (13–328) pg/ml. This is also lower than the levels observed in ADPKD patients in the current study. It should also be noted that renal function was still within the normal range in the young ADPKD subjects in the current study. Serum angiogenic growth factor levels were also significantly related to renal function. Ang-1 levels were significantly correlated with urinary protein excretion (P ¼ 0.009; Table 3). Previous animal studies have demonstrated a strong time-dependent expression of Ang-1 in glomeruli of a diabetic rat model, and correlation with glomerular volume, glomerular area, and urinary protein.25 Serum VEGF level was correlated with creatinine clearance (P ¼ 0.022; Table 3). This result indicates that serum VEGF may indicate very early renal changes as kidney function is essentially preserved in young patients with ADPKD. The mean creatinine clearance in the current study was 130.24±29.4 ml/min per 1.73 m2 (Table 1). Significant positive correlations between LVMI and Ang-1 and VEGF were also demonstrated in the current study. In particular, log10 transformed VEGF level was strongly related to LVMI after correction for age and sex (Po0.0001; Table 4, Figure 7). This is of particular relevance as patients with ADPKD are at an increased risk for left ventricular hypertrophy.26 Moreover, higher serum VEGF level in young ADPKD patients in the current study was associated with elevated LVMI even in the absence of overt hypertension. We have previously shown increased LVMI in children with ADPKD who are hypertensive or who have borderline hypertension (blood pressure between 75th and 95th percentile for age, height, and sex).5 Identification of children at risk for cardiovascular complications is important because cardiac magnetic resonance imaging and/or echocardiography are not routinely performed on young patients with ADPKD. However, future studies to investigate the role of angiogenic growth factors in the etiology of cardiac damage in ADPKD will be necessary. Previous studies have demonstrated increased plasma and platelet levels of Ang-1 and VEGF in hypertension that decreased with treatment.19 In the current study, 26 subjects were hypertensive (blood pressure X95th percentile for age, height, and sex) and 45 were normotensive. However, there Kidney International (2011) 79, 128–134
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was no effect of Ang-1 (odds ratio ¼ 0.993 (0.969–1.018), P ¼ 0.59) and VEGF (odds ratio ¼ 0.765 (0.548–1.068), P ¼ 0.1159) on the presence of hypertension. Moreover, it has previously been shown that hypertensive patients with target organ damage (stroke, myocardial infarction, angina, left ventricular hypertrophy, and mild renal failure) had higher levels of VEGF and Ang-1.20 This report agrees with the findings of our current study and indicates that both Ang-1 and VEGF may have potential roles in the early stages of renal and cardiac damage in ADPKD. In the current study, a relationship between VEGF level and Ang-1 was demonstrated. Furthermore, forward selection in multiple linear regression used to assess independent effects of Ang-1 and VEGF on all dependent variables suggest a mediation effect of VEGF on Ang-1 (Table 5). Stimulation of Ang-1 by VEGF has previously been demonstrated in bovine retinal pericytes.27 In addition, a recent study demonstrated that VEGF activates Tie-2 via proteolytic cleavage of the Tie-1 tyrosine kinase resulting in transphosphorylation and activation of Tie-2.28 This suggests a synergistic action of the angiopoietin and VEGF growth factors. However, while VEGF appeared to have a stronger relationship to renal structural parameters and LVMI, notably each growth factor had a unique relationship between specific renal functional parameters as evidenced by the relationship between VEGF and creatinine clearance and Ang-1 with urinary protein excretion. Activation of the renin–angiotensin system occurs early in ADPKD29–32 and may in part explain the upregulated expression of both VEGF and angiopoietins. In rats, increased renal expression of VEGF, Ang-1, and Ang-2 following infusion of angiotensin has previously been demonstrated.33,34 Furthermore, Ang-2 has been shown to stimulate division of human endothelial cells in culture and to upregulate expression of VEGF.35 These effects were blocked by candesartan implicating involvement of the angiotensin-1 receptor. In conclusion, VEGF level increases with expanding kidney volume and rise in LVMI, possibly reflecting a role of this growth factor in early renal and cardiac disease progression in ADPKD. Future animal and cell studies that focus on angiogenesis in ADPKD models will be required to unravel the involved molecular mechanisms, but may provide potential new therapeutic targets for ADPKD. METHODS Patient description and clinical evaluation
All patients gave informed consent to a protocol that was approved by the Colorado Multiple Institute Review Board. Patients were recruited from those participating in ongoing clinical studies of ADPKD at the University of Colorado Denver between 1990 and 2009. ADPKD diagnosis was based on radiographic imaging and Ravine’s criteria;36 all patients included in this study had a positive family history of ADPKD. All patients were studied during a 2-day in-patient visit at the Pediatric Clinical Translational Research Center at the Children’s Hospital in Denver Colorado, as described Kidney International (2011) 79, 128–134
previously.5 Serum and urine creatinine, height, and weight were used to calculate creatinine clearance for all subjects. Blood was drawn for serum chemistries and growth factor assay. Two 24 h urines were collected for urinary protein excretion and the value reported is the mean for both 24 h urines. Urinary protein was determined in the hospital core laboratory by the pyrogallol red method. Left ventricular mass was determined by standard two-dimensional and Doppler echocardiogram as previously described5 or by cardiac magnetic resonance imaging on a 1.5 T Siemens system (Siemens, Malvern, PA, USA). Cardiac-gated breathhold two dimensional true-Fast Imaging with Steady state Precession (Fast Imaging Employing Steady-state Acquisition) short-axis cine images were obtained to cover the left ventricle atrioventricular ring to apex (10 mm slice thickness, no gap, field of view 250–320 mm; 10–15 breath holds to cover the whole ventricle). The imaging protocol consisted of localizers in three orthogonal planes (axial, coronal, and sagital). After the data were acquired, a Leonardo (Siemens Medical System) workstation was used to manually contour endocardial and epicardial contours of the left ventricle in end-diastole and end-systole, and left ventricular mass was calculated. Left ventricular mass was corrected by body surface area and reported as LVMI in g/m2. Angiopoietin and VEGF assay
Patient sera were stored at –80 1C before assay and all samples were assayed in duplicate. Ang-1 and VEGF were assayed by enzyme-linked immunosorbent assay using kits from Raybiotech (Norcross, GA, USA). The intra- and inter-assay coefficient of variability of the Ang-1 assay was o10% and o12%, respectively, and lower limit of detection 30 pg/ml. The VEGF intra- and inter-assay coefficient of variability was o10 and o12%, respectively, with lower limit of detection 20 pg/ml. Serum Ang-2 was assayed by Quantikine enzyme-linked immunosorbent assay from R&D Systems (Minneapolis, MN, USA). The intra- and inter-assay reproducibility was 7 and 10.4%, respectively, with lower limit of detection, 21.3 pg/ml. Statistical analysis
All variables were checked for the distributional assumption of normality. Owing to the highly skewed distributions of all outcome variables, log10 transformations were applied and the transformed variables were used in all analyses. Log10 transformations were also applied to VEGF and the ratio of Ang-1/Ang-2. All statistical analyses were performed using SAS version 9.2 (SAS Institute, Cary, NC, USA). Descriptive statistics are presented as mean, s.d., and 95% confidence interval or geometric mean and 95% confidence interval. Univariate linear regression was used to test the relationship of VEGF, Ang-1, Ang-2, and the ratio of Ang-1/Ang-2 on ADPKD phenotypes including total renal volume, total cyst number, total cyst volume, creatinine clearance, LVMI, and urinary protein excretion. Stepwise linear regression was used with the forward selection method to determine independent 133
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effects of Ang-1, Ang-2, and VEGF on all dependent variables. No correction for multiple end points was applied; however, the primary hypothesis was that Ang-1 would be related to kidney volume measures. In linear regression, renal volume was adjusted for age, sex, and body surface area, renal cyst volume was adjusted for age and sex. Creatinine clearance was corrected for bovine serum albumin, and proteinuria was corrected for age and sex. LVMI was already adjusted for body surface area, but age and sex were included as covariates. Logistic regression was used to test the relationship of VEGF, Ang-1, Ang-2, and ratio Ang-1/Ang-2 with hypertension. In analysis of total renal volume, total cyst volume, creatinine clearance, and LVMI by quartile of VEGF; all P-values were corrected for multiple comparisons by the Tukey–Kramer correction.21
BY Reed et al.: Angiogenic growth factors and ADPKD
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DISCLOSURE
All the authors declared no competing interests. 21.
ACKNOWLEDGMENTS
This research was supported by Grant numbers M01RR00051, M01RR00069, the General Research Centers Program, National Center for Research Resources (NCRR)/NIH; by NIH/NCRR Colorado CTSI Grant number ULI RR025780, by Grant DK34039 from NIH (NIDDK), and by the Zell Family Foundation. The content of this publication is the author’s sole responsibility and does not necessarily represent the official NIH views.
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